Purpose/Objective(s) Recent studies have shown that vascular parameters of brain tumors derived from DCE-MRI may act as potential biomarkers for radiation-induced acute effects. However, accurate characterization of the spatial regions affected by radiation therapy (RT) remains challenging. Here, we introduce an unsupervised adaptive model for classification and ranking of the RT-affected regions in an animal model of cerebral U-251n tumors. Materials/Methods Twenty-three immune-compromised-RNU rats were implanted with human U251n cancer cells to form an orthotopic glioma (IACUC #1509). For each rat, 28 days after implantation, two DCE-MRI studies (Dual Gradient Echo, DGE, FOV: 32 × 32 mm2, TR/(TE1-TE2) = 24 ms/(2 ms-4 ms), flip angle = 18°, 400 acquisitions, 1.55 sec interval with Magnevist contrast agent, CA injection at ∼ 24 sec) were performed 24h apart using a 7T MRI scanner. A single 20 Gy stereotactic radiation exposure was performed before the second MRI, which was acquired 1-6.5 hrs after RT. DCE-MRI analysis was done using a model selection technique to distinguish three different brain regions as follows: Normal vasculature (Model 1: No leakage, only plasma volume, vp, is estimated), leaky tumor tissues with no back-flux to the vasculature (Model 2: vp and forward volumetric transfer constant, Ktrans, are estimated), and leaky tumor tissues with back-flux (Model 3: vp, Ktrans, and interstitial volume fraction, ve, are estimated). Normalized time traces of DCE-MRI information (24 pre, and 24 post-RT for each rat, total of 64108 training datasets) of tumors and their soft surrounding normal tissues were extracted from the 3 different model regions. To eliminate high-dimensional data similarity, an unsupervised autoencoder (AE) was trained to map out the model-derived data into a feature space (latent variables, N=10). For each model, the pre and post RT latent variables were compared (by appropriate tests of significance: ANOVA/Welch, CI=95%) to reveal RT-discriminant features. Pearson correlation coefficients were used to compare the decoded data to rank the effect of RT on different models. Results The time trace of DCE-MRI information of rat brain in normal (Model 1, non-leaky) and highly permeable (Model 3) regions are less impacted by RT (Higher correlation between pre and post RT: r= 0.8518, p<0.0001 and r= 0.9040, p<0.0001 for Model 1 and Model 3, respectively) compared to the peritumoral regions pertaining to Model 2 (r= 0.8077, p<0.0001). Conclusion This pilot study suggests that among different brain regions, peritumoral zones (infiltrative tumor borders with enhanced rim) are highly affected by RT. Spatial assessment of RT-affected brain regions can play a key role in optimization of treatment planning in cancer patients, but presents a challenging task in conventional DCE-MRI. This study represents an important step toward classification and ranking the RT-affected brain spatial regions according to their vascular response following hypofractionated RT.

Purpose/Objective(s)Triple negative breast cancer (TNBC) is the most aggressive breast cancer (BC) form, with a high metastases rate and a very low survival. The aggressiveness of TNBC coupled with a significant toxicity and suboptimal chemotherapy outcomes underscores the urgency for new TNBC treatments. In recent years, immunotherapy has emerged as a promising option. In particular, immune checkpoint blockers (ICB) targeting PD-L1/PD1 inhibitory T cell check point pathway showed clinical responses and have been explored for TNBC. Unfortunately, the response rates to standalone ICB therapy are low (15-20%), indicating the presence of inhibitory immune mechanisms. Radiation therapy (RT) has been widely used in BC therapies. In addition to antitumor (antiproliferative) effects, RT has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME) that has been shown to enhance the response to immunotherapy in mouse BC models. Antitumor RT effects, including TME immunomodulation, can be improved by using radiosensitizers, such as gold nanoparticles (AuNPs). We hypothesize that AuNP potentiates RT-induced immunomodulatory effects, leading to a more efficient response to ICB in TNBC. To test this hypothesis, we used AuNP as an enhancer of RT-induced immunological TME changes, to improve ICB therapy response in murine orthotopic syngeneic 4T1Luc TNBC model.Materials/MethodsFemale Balb/c mice bearing 4T1Luc tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with fractionated regimen of 3 × 6 Gy dose using 225 kV photons. After the 3rd RT dose, mice received 3 doses of anti-PD-L1 antibody that were 4 days apart. Therapeutic efficiency was determined by assessing the tumor growth and animal survival. Tumor tissue immunohistochemistry determined the expression of TME immunological markers and immune cell tumor infiltration.ResultsAuNPs improved response to anti PD-L1 treatment in mice receiving RT, shown by significant delay in tumor growth and increase in survival compared to the animals receiving RT+ AuNP (p<0.01) and to the animals receiving RT+ anti PD-L1 or RT alone (p<0.05). These results were accompanied with changes in the expression of TME immunological markers and T cell and macrophage infiltration.ConclusionIn TNBC patients, induction of antitumor immune response may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the effect of a fractionated RT regimen that has significantly improved the response to anti PD-L1 treatment in 4T1Luc TNBC mouse model. This effect was measured by a delay in tumor growth and an increase in animal survival. These findings support the role of immunological mechanisms in TNBC and provide a platform for designing multimodal TNBC RT formulations with novel radiosensitizers or immunotherapy.

In recent years, radiation therapy (RT) has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME). One of the RT immunomodulation mechanisms include immunogenic cell death (ICD) that plays a major role in stimulating host anti-cancer immune response and can determine the success of cancer RT. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Calreticulin (CRT) is a DAMPs molecule involved in phagocytosis and dendritic cell antigen presentation. In breast cancer (BC) calreticulin pronounced expression was associated with tumor metastatic potential and size. Therefore, DAMPs are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BC therapies, the effect of RT and radiosensitizers (such as gold nanoparticles (AuNP)) on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CRT expression and associated macrophage infiltration in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BC mouse models. We hypothesize that AuNP modulates RT induced immunological changes such as increase in CRT expression and infiltration by F4/80 positive macrophages. Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 4nm or 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CRT and anti F4/80 antibodies and analyzed by light microscopy. In animals receiving RT or 14 nm AuNP only, CRT and F4/80 expression exhibited trend in increase relative to control but did not reach the significance. However, after the combined RT and AuNP (4nm or 14nm) treatment, CRT expression was further increased and reached the significance, compared to controls. However, F4/80 expression was significantly increased only in animals receiving the combination of RT and 14 nm AuNP, compared to controls (Table 1). In BC patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single RT dose in BC mouse model. This effect was measured by an increase in the expression of CRT and F4/80, an indicator of macrophage infiltration. These findings support the role of immunological mechanisms in BC depends and provide a platform for designing multimodal BC RT formulations with novel radiosensitizers or immunotherapy.Abstract 3252; Table% positive staincontrolRT4 nm AuNP14 nm AuNPRT + 4 nm AuNPRT + 14 nm AuNPCalreticulin3.549.714.5612.6219.22*17.42*F4/8019.4422.4917.4520.4721.2030.99**p<0.05 compared to control. Open table in a new tab

Gold nanoparticles (AuNPs) are explored as radiosensitization probes enhancing cancer radiation treatment effects. Our long range goal is to fully characterize and optimize parameters for AuNPs use in cancer radiotherapy. We aim to develop AuNP probes that can be delivered to the cancer cells with the purpose of increasing their radiation sensitivity, and that would facilitate targeted delivery of high radiation doses to cancer, while sparing surrounding normal tissue from radiation toxic side effects.

The current study evaluates dosimetric and spectral effects when platinum (Pt)-based chemotherapeutics and less toxic tungstophosphoric-acid (TPA) organometallics are present during x-ray radiotherapy. We hypothesize that the use of high energy photon beams (i.e. 18 MV) will increase absorbed dose due to increased pair production from high-Z elements and thus result in additional tumor cell kill. EGSnrc code was used to examine the contribution of pair production to dose in the presence of the high-Z material (TPA, Pt mixtures and tungsten, W) as a function of beam energy. Variables included different concentrations (100 µmolar, 1 mmolar), depths (5 mm, 10 cm), thicknesses (5 mm, 5 cm) and energies (6, 18 MV). Overall, for the deeper depth, the 511 keV photon fluence increase was up 31% (18 MV-1 mmolar) while at 6 MV it was between 10%-11% depending on the concentration. For the shallower depth, 18 MV fluence increase was up 14.6% (1 mmolar) and 18.6% (1 mmolar) for the 6 MV. The dose enhancement effect due to pair production was up 25%-30% and a total 33%-58% depending on the depth. The benefit related to pair production was more for 18 MV and under conditions that simulated a realistic clinical setup. While part of the effect could be attributed to photoabsorption, a significant contribution of dose could result from pair production. Experimental clonogenic survival assay was consistent with the theory in that the low dose shoulder region of a cell survival curve was reduced using TPA and 18 MV compared with TPA and 6 MV or compared with no TPA and 18 MV; RBE was approximately 2 at the dose commonly used in conventional fractionated clinical radiotherapy. This suggests a potential new strategy for dose enhancement based on pair production using higher energy beamlines.

To perform radiomics analyses on normal lung tissue delineated from CT-based image datasets for patients with locally advanced, non-small cell lung cancers (NSCLC) in order to characterize differences between patients with and without radiation-induced pneumonitis (RP). Planning and 3-month follow-up CT image datasets of forty-one patients (14 with RP and 27 with no evidence of RP) with stage-III lung cancers, treated with IMRT/3D-CRT, were investigated. One hundred sixty-eight radiomics features were extracted from the volume of normal lung tissue receiving ≥20 Gy, excluding the ITV, according to the following 8 different classes: Intensity Histogram Based Features (IHBF), Gray Level Run Length (GLRL), Law’s Textural information (LAWS), Discrete Orthonormal Stockwell Transform (DOST), Local Binary Pattern (LBP), Two-Dimensional Wavelet Transform (2DWT), Two Dimensional Gabor Filter (2DGF), and Gray Level Co-Occurrence Matrix (GLCM). Analysis of variance (ANOVA) was used to compare differences between radiomics features. Fisher's-combined method with mean-percent-difference (MPD) measures was used to compare CT-based radiomics features between non-RP and RP patients. Among 168 radiomics features compared on planning CT images, 2 were significantly different between RP and non-RP groups: Intensity-Based-Histogram-Feature (IBHF, entropy): p=0.0334, MPD=-27.71% and 2D-Wavelet-Transform (2DWT, entropy): p=0.0214, and MPD= 185.59%. For patients with RP, 6 features were significantly different, with pFisher<0.001 for all categories: IBHF, DOST, and DTW, between RP grades 1 and 2 sub-groups. On follow-up CT images, 62 features were significantly different between RP and non-RP groups, with pFisher<0.001, in all categories: IBHF (MPD=57.78%), Gray-Level-Run-Length (MPD=28.61%), LAWS (MPD=-15.74%), Discrete-Orthonormal-Stockwell-Transform (MPD=13.37%), Local-Binary-Pattern (MPD=30.33%), 2D-Wavelet-Transform (MPD=37.02%), 2D-Gabor-Filter (MPD=-3.08%), and Gray-Level-Co-occurrence-Matrix: (MPD=-30.83%). Eighteen features were significantly different between RP grades 1 and 2 sub-groups, with pFisher<0.001 in six categories: IBHF, GLRL, LAWS, DOST, 2DWT, GLCM. Results suggest that the entropy of normal lung tissue, associated with the arrangement of intensities (IBHF) and frequency homogeneity (2DWT) on planning CT datasets demonstrate promising biomarkers for development of radiation-induced pneumonitis. High-frequency image components were found to be significantly impacted by RT between RP and non-RP CT datasets. The results of this pilot study, albeit subject to confirmation in a larger patient population, suggest a potential role for the use of radiomics-based signatures in models developed for predicting radiation-induced lung damage in patients with locally advanced-NSCLC.

Of all human cancers, lung cancer remains one of the most pervasive and difficult to treat. Current treatment regimens generally involve combinations of chemotherapy, radiotherapy, and/or surgical removal of the tumor. The radiomodulator, nano-genistein, may mitigate the toxicities associated with radiation therapy when administered to patients being treated with chemoradiotherapy for non-small cell lung cancer (NSCLC). The objective of the ongoing phase 1b/2a trial described here is to evaluate the pharmacokinetics (PK), safety, and efficacy of nano-genistein and concurrent chemoradiotherapy for NSCLC treatment. Patients with newly diagnosed stage II, III or IV NSCLC for whom 60 Gy radiation therapy and weekly paclitaxel/carboplatin is the recommended therapy are eligible. Patients are treated daily with nano-genistein at one of three dose levels (500 mg, 1000 mg, or 1500 mg) starting prior to and continuing during the entire course of concurrent chemoradiotherapy (up to 8 weeks). The three cohorts will be enrolled sequentially, each enrolling a minimum of 6 patients. PK analysis will be completed for nano-genistein, paclitaxel and carboplatin. Dose-limiting toxicities (DLTs) are defined as related to study drug and Grade 4 radiation esophagitis or Grade 3 (> 7 consecutive days), Grade 4 neutropenia (> 7 days), thrombocytopenia (> 7 days), or nausea/vomiting, Grade 3 thrombocytopenia with clinically significant bleeding, AST/ALT elevations > 7 days or non-hematologic toxicities Grade 3 or higher, in addition to delays in radiotherapy or chemotherapy due to toxicity of more than 3 weeks. Response and pneumonitis are measured using CT scans and clinical assessment. Safety is assessed using vital sign measurements, physical examinations, and diagnostic measurements (clinical chemistry, hematology, urinalysis, and ECGs) and adverse events are reported using the NCI Common Terminology Criteria for Adverse Events (CTCAE). Quality of life measurements include the University of California, San Diego Shortness of Breath questionnaire, Functional Assessment of Cancer Therapy - Lung Trial Outcome Index and a swallowing diary. To date, the study has enrolled 13 patients; 7 in Cohort I and 6 in Cohort II. Subjects presented at a median age of 72 years; 4 males, 9 females; 4 with Stage II disease and 9 with Stage III; 7 with adenocarcinoma and 6 with squamous cell histology. Safety, PK, and effects of nano-genistein mediated reduction on radiation toxicity and pharmacodynamic biomarkers will be assessed during patient follow up and will be reported at the time of study completion. The data from this trial will be used to select the dose for an adequately powered, randomized, double-blind, placebo-controlled phase 2b study of nano-genistein in NSCLC patients.

To enhance radiotherapy by utilizing pair-production, the probability of which increases with X-ray energy and atomic Z number. Introducing high Z metals during 18MV irradiation should result in increased cell killing compared to 18MV irradiation without metal or 6MV with/without metal. This effect will be due to increased pair-production near high Z nuclei which increases locally absorbed radiation dose. This effect requires metal, preferably nontoxic, to be present during irradiation and will be measured in vitro with clonogenic survival assays. Clonogenic survival assays were carried out using A549 human lung cancer cells. Tissue culture plates were irradiated with 2Gy of either 6MV or 18MV photons with or without metal drugs (100 uM sodium phosphotungstate hydrate, 4ug/ml cisplain, and 12ug/ml carboplatin) present during radiotherapy. Metal drugs were introduced to culture media 2 hours prior to irradiation and left in place during irradiation. The media was replaced immediately after irradiation with metal-free media. Calibrated output factors were used to ensure the same absorbed dose between the two beam energies under identical experimental setup conditions. Mean plating efficiency was determined to be 41% on average for A549. Incubating cells in 100uM tungsten for 4 hours without irradiation produced no significant toxicity (>95% survival). In the absence of metal drug, the 2Gy 6MV and 18MV plates showed similar survival (64% vs 67%). When tungsten compound was introduced, the 6MV plate again showed 66% survival, however the 18MV plate showed 34% survival, approximately double cell killing. These experiments were repeated at 4, 6, and 8Gy to generate cell survival curves and the radiation sensitization enhancement ratio (SER) was found to be approximately 1.5. The tungsten 18MV survival curve shows a minimal shoulder region compared to the 6MV with/without tungsten and 18MV without tungsten curves, suggesting inhibition of sublethal damage repair. The 2Gy 6MV vs 18MV clonogenic survival studies were carried out for 4ug/ml cisplatin and 12ug/ml carboplatin which similarly showed approximately 40% enhancement of cell killing for both drugs at 18MV compared to 6MV. Cells treated with 18MV irradiation in the presence of 100uM sodium phosphotungstate hydrate showed approximately 50% less cell survival at 2, 4, 6, and 8Gy compared to 6MV with/without metal and 18MV without metal. Cisplatin and carboplatin show similar radiosensitization at 18MV yielding 40% more cell killing than 6MV. This effect is due to pair-production leading to increased photon-matter interaction and requires metal to be present at time of irradiation. The survival curves at 2, 4, 6, and 8Gy show loss of initial shoulder region suggesting impaired sublethal damage repair or increased effective LET. The generation of 511keV annihilation photons in situ may allow tumor imaging and dose measurement via PET technology. Confirmatory in vivo studies are planned.

Metformin produces direct cancer stem/non-stem cell cytotoxicity, inhibits tumor growth, protects normal tissues from various stressors, and sensitizes cancer stem/non-stem cells to chemo and radiation therapy. However, the mechanism governing metformin’s anti-cancer activity is not fully understood. This study investigates metformin’s biochemical effect on cancerous and non-cancerous cells under varying environmental conditions using reactive oxygen species (ROS) as a marker of cytotoxicity, and compares metformin’s effect on cancer stem vs non-stem cell ROS levels. A549 (lung cancer), MCF-7 (breast cancer), and MCF-10A (normal breast) cell lines were exposed to 5mM metformin for 4-6 hours in media containing variable glucose concentration, pH, and oxygen levels. ROS production was measured using dihydroethidium (DHE) fluorescence reagent with flow cytometry spectroscopy. MCF-7 stem and non-stem cells were identified using FITC-CD44 and APC-CD24 antibody FACS. Clonogenic and MTT assays were used to assess cytotoxic cellular response. Metformin significantly increased ROS in A549 and MCF-7 cell lines, while the benign MCF-10A cell line was not affected. A549 cells treated with metformin in 4.5g/L glucose DMEM showed a 139% ROS increase compared to control cells without metformin. A549 in 0g/L glucose free medium (GFM) with metformin had ROS levels 203% higher compared to no drug. Hypoxia and altered pH did not significantly change metformin’s effect on A549 ROS at either glucose level. MCF-7 cells in 4.5g/L glucose DMEM treated with metformin showed a 59% ROS increase compared to control. MCF-7 with metformin in GFM showed 154% increase in ROS levels compared to no drug. Cytotoxicity and radiosensitization was observed in A549 treated with metformin using clonogenic assay, and with MCF-7 using clonogenic and MTT assays. No radiosensitization or cytoxicity was observed in MCF-10A cells exposed to metformin. CD44+high/CD24-low stem cell populations of MCF-7 showed baseline ROS levels 300% lower than CD44+high/CD24+highnon-stem cell populations. Metformin exerted equal effect on both stem and non-stem cell population ROS levels. Metformin increases ROS levels in A549 and MCF-7 cancer cells via a [glucose] dependent mechanism which translates into cytotoxicity. Benign MCF-10A cells are not significantly affected. Metformin’s effect on ROS levels of cancer cells, particularly cancer stem cells, may provide some explanation for the decreased incidence of malignancy and improved outcomes in diabetic patients on metformin. Interestingly, this work suggests that metformin may be even more beneficial in non-diabetic patients with lower blood glucose levels. Further studies are underway in hope of translating these findings into clinical practice.

The purpose of this study was to explore the relationship between tumor interstitial fluid pressure, which has historically been used as a measure of tumor aggressiveness, and expression of CD44, a marker of cancer stem cells. Cancer stem cells (CSCs) are a subpopulation of tumor cells that express specific proteins that putatively confer an aggressive proliferative, invasive, and metastatic phenotype such that CSCs have the ability to initiate the regrowth of a tumor. One accepted marker of CSCs is CD44, the receptor for hyaluronic acid (HA). CD44 has been shown to be overexpressed in several cancers, including primary cancers from breast, prostate, lung, pancreas, brain, and from metastatic brain tumors. Tumor aggressiveness has been historically measured invasively using tumor interstitial fluid pressure (TIFP). It has been demonstrated theoretically that TIFP can be measured non-invasively using contrast enhance magnetic resonance imaging (CE-MRI). Recently, it was demonstrated experimentally that both interstitial fluid flux and tissue porosity could be estimated by CE-MRI, thus potentially leading to the development of a non-invasive estimate of TIFP. The objectives of the study were to further validate CE-MRI as a non-invasive tool to measure tumor aggressiveness by determining the correlation between CD44 expression and a non-invasive MRI TIFP measurement. Transplanted U251 human glioblastomas were grown intracranially in nine athymic rats. When the tumors reached between 3 and 6 mm in maximum diameter, TIFP was measured non-invasively using CE-MRI. TIFP was measured in units of mmHg. At the completion of the TIFP measurement, tumors were excised, formalin fixed and processed for CD44 expression. CD44 was semi-quantitatively scored following the procedure of the Allred IHC scoring system. There was a positive significant correlation between CD44 expression and the non-invasive MRI TIFP measurement (R squared = 0.68). A possible explanation is that CD44 is upregulated in response to increased physical stress as a consequence of the high TIFP. The two parameters, TIFP and CD44 were directly and positively correlated. The predictive value of TIFP to measure CD44 expression should be further explored. Future work will incorporate other tumor types and response to therapies, especially radiation therapy.

Purpose Treatment of uveal melanoma can impair patients’ psychological well-being. We evaluated patient-reported outcome measures (PROMs) of anxiety, depression, and quality of life (QoL) over 2 years following treatment in a consecutive sample of uveal melanoma patients, compared observations to population normative values and examined whether outcomes differed according to patients’ age, gender, and whether or not they were treated by enucleation or had a poor prognosis (presence of monosomy 3). Design Prospective longitudinal study. Participants Patients ( N =411) with uveal melanoma treated between 2008 and 2011. Methods Self-report questionnaire study. We compared mean PROMs scores obtained 6 months, 1 year, and 2 years after treatment to published population normative values using 2-sample t -tests, and tested the association of these scores with gender, age, treatment by enucleation, and monosomy 3 using mixed-model ANOVAs. Results On QoL and depression, patients were similar to or better than normative values at all time points, but there was some evidence that females were more anxious than female normative values (Ps<0.001–<0.05). Younger patients ( P <0.01) and female patients ( P <0.01) were the most anxious overall. Enucleation was not associated with PROMs. Patients with monosomy 3 showed more depressed mood at all the three time points ( P <0.05). Conclusions Patients treated for uveal melanoma can expect, within 6 months of treatment, to have a QoL that is similar to that of the general population. Younger female patients and patients with monosomy 3 are more likely to be distressed, and clinicians will need to be alert to this.

To investigate the correlation between pathology and Raman spectroscopic signal from tumor and normal tissue after radiation exposure in an animal model. Nineteen female Balb/C mice were investigated. Five mice were used as “unirradiated normal lung” controls. 14 mice were implanted with 4T1 breast cancer cells (∼1/2 million) to their right flanks. 4T1 breast cancer cells injected intramuscularly metastasize to the lungs by day 14. Among these 14 mice, 5 were “unirradiated tumor” controls. After day 14, the remaining 9 mice received radiation in a single fraction of 12 or 18 Gy to the left lung with 6MV photons. One to two days after radiation, lungs were removed, snap frozen, and stored in a -800C freezer. Lung specimens were sectioned using a cryostat-microtome for pathologic evaluations and Raman measurements. Principal component analysis (PCA) and discriminant function analysis (DFA) were used to analyze the data. Table 1 shows the results of the PCA/DFA analyses relative to pathologic scoring as well as the number of Raman spectra recorded from each group. A total of 675 Raman spectra were collected as follows: 126 (unirradiated tumors), 108 (tumors receiving 12 Gyx1), 130 (tumors receiving 18 Gyx1), 204 (irradiated normal lung tissues (bearing tumors)), with 91 of these normal lung spectra from 12 Gyx1, and 113 from 18 Gyx1; 107 (unirradiated normal lung tissues). Raman spectroscopy was able to differentiate unirradiated tumor and unirradiated normal tissues with 100% sensitivity relative to pathologic scoring. The classification results showed an overall accuracy of 81.9% relative to pathology evaluation in identifying unirradiated tumor vs. irradiated tumors with 12 or 18 Gy. Similarly, Raman spectroscopy was able to differentiate unirradiated normal from irradiated normal lung tissues with a specificity of 87.9%. Raman spectroscopy is shown to identify irradiated vs. unirradiated lung tumors and normal lung radiation response with high sensitivity and specificity relative to pathologic scoring. This suggests a role for the Raman method to diagnosis the presence of tumor and in analyzing post-radiation patient tissues to assess response.Poster Viewing Abstracts 3496; Table 1A summary of PCA/DFA classification of each group relative to pathological evaluationDescriptionGroupNo. of Raman spectraClassification relative to pathologic scoringNormal vs. TumorUnirradiated normal lung107100%Unirradiated lung tumor126100%Unirradiated vs. irradiated normal lungUnirradiated lung tumor12678.512 Gy irradiated lung tumor10885.718 Gy irradiated lung tumor13081.4Unirradiated vs. irradiated normal lungUnirradiated normal lung10787.912 Gy irradiated normal lung9187.918 Gy irradiated normal lung11383.2 Open table in a new tab

Deformational and mass changes associated with regression of the visible tumor during the course of fractionated radiotherapy have confounded the ability to perform accurate deformable image registration, subsequently limiting the clinical implementation of adaptive radiotherapy. This study sought to investigate the impact of tumor regression on the accuracy of deformable image registrations (DIR) and then to find a solution to improve the performance of DIR for treatment of lung cancer patients. Specifically, daily cone-beam computed tomography (CBCT) images were acquired from three locally advanced NSCLC patients. DIRs were performed from fractions 1, 10, and 20 to fraction 25 using a B-Spline-based algorithm implemented within the VelocityAI platform. To improve the accuracy of the BSpline- based registrations in the region of regressing tumors, a hybrid finite element method (FEM) was developed with a mesh defined in a bounding box surrounding the tumor in the target image. The constraints of the FEM model were derived from the displacements generated by the B-Spline registrations. Using the displacement vector fields (DVFs) of the B-Spline and hybrid registrations, the source images were warped to their targets. The accuracies of the two registration algorithms were evaluated, using landmark points identified on both the source and target images, as well as quantitative analysis of the generated DVFs. For the three patients, average tumor volumes were reduced by 53 fraction 1 and fraction 25. Comparison of landmark points showed that the mean errors of the FEM-based hybrid registrations were 1.4, 1.6, and 1.7 mm for the three patients. The average displacement differences between the B-Spline and FEM-hybrid registrations for the three patients were 4.8, 6.2 and 3.9 mm with a maximum of 15 mm for patient 2. Lung tissue does not move consistently with the shrinking tumor. The more the tumor regresses, the larger the B-Spline registration error in the tumor region. The proposed hybrid method that consists of the intensitybased image registration and mechanics-based tissue modeling to correct geometric changes induced by anatomical deformation and tumor regression, respectively, may have the potential to improve the quality of adaptive radiation therapy for lung cancer patients.

The purpose of this study is to explore whether Raman spectroscopy is able to assess the response of lung tumors and healthy lung tissue in mice following radiation therapy. 4T1 mouse breast cancer cells injected intramuscularly metastasize to the lungs at day 14. These 4T1 cancer cells were injected subcutaneously into the flanks of 18 Balb/C female mice. Five additional mice were used as “normal lung” controls. After 14 days, cohorts of mice bearing tumors received 6, 12 or 18 Gy to the left lung with 6MV photons. Five mice were treated as “unirradiated tumor” controls. After 24- 48 hours, lungs were excised and the specimens were sectioned for Raman measurements and pathologic evaluation using a cryostat-microtome. A total of 775 Raman spectra were collected; 107 from unirradiated normal lung tissues, 126 from unirradiated tumors, and 318 from tumors irradiated with 6, 12 or 18 Gy. Raman spectra were also collected from normal lung tissues of mice with unirradiated tumors (29) as well as irradiated (6, 12 or 18 Gy) tumors (195). Principal component analysis (PCA) and discriminant function analysis (DFA) were performed to analyze and interpret the results. Normal lung tissues and tumors were identified 100% of the time relative to pathologic scoring. Raman spectral data showed prominent results between unirradiated tumor and tumors receiving 12 or 18 Gy. Thus, in a model consisting of unirradiated and irradiated tumors (12 or 18 Gy) classification accuracies were 97.6%, 79.6%, and 80.8%, respectively, relative to pathologic assessment. Overall, 85.4% distinguishability was observed for unirradiated and irradiated (6, 12 or 18 Gy) normal lung tissues. Preliminary results demonstrate the promise for Raman spectroscopy in the prediction of normal vs. lung tumors as well as in the assessment of response of tumor and normal lung tissues following radiation therapy.

To determine specific tumor biomarkers predicting radiation therapy response in non-small cell lung cancer (NSCLC) patients. Tumors from NSCLC patients (n = 133) treated with curative radiation therapy alone or combined with chemotherapy (initially presenting with stage I, II or III disease comprising 21%, 15% and 64% of patients, respectively) were examined for the following 8 biomarkers: ALDH1A1, CD68, Hyaluronic acid, Beta-Catenin, CD44, MFG-E8, Matrix metalloproteinase-9 (MMP-9) and Vimentin, as a control. The markers were selected based on their correlation with TGF-beta expression and/or association with tumor aggressiveness, radioresistance and poor prognosis in several tumor indications. Survival time was calculated using a Cox proportional hazards model. Univariate analyses followed by multivariate models were examined to determine biomarkers and other factors predictive of survival. A logistic regression model was used to analyze factors predicting for local control. High CD68 expression was associated with an increased risk of death (p = 0.008), while there was a trend towards a decreased risk of death associated with high MMP-9 expression (p = 0.05). Squamous cell carcinoma histology was associated with higher -odds of local failure compared to adenocarcinoma [Odds ratio, OR = 4.39, 95% confidence interval, CI = (0.96, 19.98)]. Expression of CD44 was associated with significantly increased odds of local tumor failure [OR = 1.44, 95% CI = (1.08, 1.92)], while MFG-E8 was associated with decreased odds of local tumor failure [OR = 0.77, 95% CI = (0.62, 0.97)]. Combined, CD44, MFG-E8, and tumor type were predictive of local control with an area under the curve value of 0.74 (1.00 being a perfect predictor). The median survival time among all patients was 1.5 years. The only patient characteristic on univariate model analyses exhibiting a difference in survival was race, with African-Americans (AA) having a 1.5 times greater risk of death than non-AA (p = 0.044), while the overall effect of race on survival was not significant (p = 0.76). The overall main effect of median household income remained insignificant (p = 0.099), as in the univariate analysis. However, after checking for effect modification, the median household income significantly modified the effect of race on survival (p = 0.037), and that model fit was improved. AA were only at a significantly higher risk when median household income was between $30,000 and $50,000 (p = 0.002). Among the NSCLC treated with radiation therapy, CD68 was predictive of increased risk of death, while MMP-9 decreased the risk of death. Racial disparities in survival were dependent on median household income. High expression of CD44 and low expression of MFG-E8 were predictive of local tumor control.

INTRODUCTION:Although controversial, use of contralateral risk-reducing mastectomy (CRRM) is increasing. It is not clear whether reduction of objective breast cancer risk or other factors determine decisions for CRRM. We aimed to identify factors that influence these decisions by scrutinising how decisions were made in one centre.METHODS:We reviewed a consecutive series of 60 patients considered for CRRM in one centre. Data sources, analysed using qualitative methods, were records of routine psychological assessment, surgeon letters, case-notes and interviews with four surgeons.RESULTS:Perceptions of objective risk did not generally drive patients' requests or surgeons' decisions. Instead, CRRM appeared to be mainly performed for psychological reasons: to reduce patients' cancer worry and to achieve cosmetic benefits.CONCLUSION:Routine use of the term 'risk-reducing' surgery masks a clinical decision which usually reflects influences other than risk. As CRRM is often carried out for psychological reasons it follows that evidence about its psychosocial outcomes is needed.

The anti-angiogenic therapy (AAT), Cilengitide (EMD121774), a cyclic RGD-motif containing peptide binding with high specificity to αvß3 and αvß5 receptors, demonstrates a small non-curative response in cerebral gliomas of rats and humans. Here we test whether 1) the timing and sequence of drug and radiation affect tumor response and 2) whether the MRI measure of vascular permeability was predictive of response to therapy. Male CD1nu/nu rats implanted with U251n cells (2x105 cells/0.1 ml) intracerebrally and stereotactically, 2-weeks before AAT was given once (4 mg/kg IP) either 1, 2, 4, 8 or 12h before or after radiation therapy (RT). RT alone and drug alone controls were included. Rats were stereotactically irradiated using a single 6 mm collimated A-P beam of 6 MV x-rays so as to deliver a central axis dose of 20 Gy was delivered to the tumor volume (95-100% isodose). Rat survival was monitored. In a separate group of rats with 2-week old tumors, MRI was performed using a 7T magnet, Bruker Avance console, and Bruker-supplied RF coils. To assess permeability, a T-One by Multiple Read-Out Pulses (TOMROP) sequence measured R1 (= 1/T1) at baseline and every 145 s following injection of a contrast agent, Gadomer (Schering AG). Using a standard kinetic theory approach, the transvascular forward transfer constant K1 (a known measure of vascular permeability) and other vascular parameters were determined. Increased Kaplan-Meier survival was evident with combined AAT and RT. When RT preceded drug, there was no survival advantage. When RT followed drug, the interval between drug and RT had a large effect on efficacy; an interval of 4 or 8 h was optimum with 100% complete response (n = 8 per group). When AAT preceded RT by less than 4 h, efficacy decreased such that at 1h, survival was only slightly better than RT alone. K1 was measured in rats with 2-week tumors given sub-curative and curative treatments as follows: rats were imaged and then given AAT (4 mg/kg, i.p.). After an interval of either 1h (sub-curative) or 8h (curative), the animals were irradiated (20 Gy) and imaged at intervals of 12 (n = 13), 36 (n = 8), or 84 (n = 8) h after RT. In the "curative" group, a more than 2-fold decrease in K1 was observed at 12 h post-RT, (from 2.3x10−3 to 1.1x10−3, p < 0.05), demonstrating AAT + RT acutely decreases tumor vascular permeability. In the "sub-curative" group, the change in K1 was small and not significant (from 2.5x10−3 to 2.1x10−3, p >0.05). Vascular permeability is a discriminant marker predictive of the success of combined radiation and antiangiogenic therapy. Cilengitide as an adjuvant to RT is currently being evaluated in clinical trials.

Viral vector mediated suicide gene therapy (SGT) involving thymidine kinase (TK) or cytosine deaminase (CD) have considerable promise in the treatment of malignant brain tumors. An unresolved issue is to what extent tumor hypoxia influences the outcome of SGT since brain tumors characterized by regions of hypoxia have potentially reduced cellular metabolism and SGT's cytotoxicity is manifest through cellular metabolism. We studied in vitro and in vivo, the effect of hypoxia on the cytotoxicity of SGT in rat 9L glioma cells. Neither acute nor chronic hypoxia affected the cell killing of SGT by TK or CD. In vivo confirmation that SGT efficacy was not adversely affected by tumor hypoxia using the hypoxic cell marker pimonidazole was shown by the absence of a change in tumor hypoxia by SGT. These studies support the use of SGT utilizing either TK or CD gene strategies even when tumors are characterized by a hypoxic microenvironment.

Vasculature in and around the cerebral tumor exhibits a wide range of permeabilities, from normal capillaries with essentially no blood-brain barrier (BBB) leakage to a tumor vasculature that freely passes even such large molecules as albumin. In measuring BBB permeability by magnetic resonance imaging (MRI), various contrast agents, sampling intervals, and contrast distribution models can be selected, each with its effect on the measurement's outcome. Using Gadomer, a large paramagnetic contrast agent, and MRI measures of T(1) over a 25-min period, BBB permeability was estimated in 15 Fischer rats with day-16 9L cerebral gliomas. Three vascular models were developed: (1) impermeable (normal BBB); (2) moderate influx (leakage without efflux); and (3) fast leakage with bidirectional exchange. For data analysis, these form nested models. Model 1 estimates only vascular plasma volume, v(D), Model 2 (the Patlak graphical approach) v(D) and the influx transfer constant K(i). Model 3 estimates v(D), K(i), and the reverse transfer constant, k(b), through which the extravascular distribution space, v(e), is calculated. For this contrast agent and experimental duration, Model 3 proved the best model, yielding the following central tumor means (+/-s.d.; n = 15): v(D) = 0.07 +/- 0.03 for K(i) = 0.0105 +/- 0.005 min(-1) and v(e) = 0.10 +/- 0.04. Model 2 K(i) estimates were approximately 30% of Model 3, but highly correlated (r = 0.80, P < 0.0003). Sizable inhomogeneity in v(D), K(i), and k(b) appeared within each tumor. We conclude that employing nested models enables accurate assessment of transfer constants among areas where BBB permeability, contrast agent distribution volumes, and signal-to-noise vary.